PROJECT SUMMARY The function of the nervous system depends on precise connections among neighboring cells. These include not only synaptic connections among neurons, but also contacts between neurons and glia. Indeed, impaired neuron-glia relationships contribute to a variety of neurodevelopmental and neurodegenerative diseases. To fulfill many of their functions, glial cells communicate with neurons through direct neuron-glia contacts with defined morphologies. However, the cellular and molecular mechanisms by which these contacts arise remain largely unexplored, except in the special case of myelination, due to the technical challenge of visualizing individual cell-cell contacts in complex nervous systems. I will overcome this barrier by using an innovative model of neuron-glia contacts in C. elegans to define the developmental steps, cellular requirements, and molecular pathways through which neurons and non-myelinating glia form specialized contacts. In particular, I will investigate how a gas-sensing neuron called BAG adheres to and precisely wraps a portion of its glial neighbor. This remarkable interaction was previously described by EM, but had not been accessible using optical approaches and so had not been further studied. We have recently developed methods for high-resolution imaging of this neuron-glia contact and have also identified molecules that likely function in BAG-glia contact development. In Aim 1, I will use quantitative imaging approaches to track the formation of the BAG-glia contact in vivo across time. I will then manipulate the system to probe how BAG develops the correct morphology at the neuron-glia interface. These experiments will test the hypothesis that the BAG-glia contact forms in a stepwise fashion, whereby the two cells adhere to one another and the glial cell subsequently instructs changes in neuronal morphology to generate the structure of the mature contact. In Aim 2, I will place the molecular factors we have identified into genetic pathways and probe how these factors affect the localization of a cell adhesion molecule, SAX-7, required for BAG-ILso adhesion. I hypothesize that at least some of the factors we have identified function in adhesion pathways independent of and in parallel to SAX-7, while other factors act by localizing SAX-7 to the site of neuron-glia contact. Importantly, all genes previously isolated in our screens are shared with mammals, suggesting that the cellular mechanisms and molecular pathways I identify are likely to be broadly conserved. Collectively, the proposed work will elucidate general principles of neuron-glia contact formation and will thereby enhance our understanding of neural function and neurological disease.